Carol E. Schrader Regulation of Antibody Diversity by AP Endonucleases Project Summary Error-prone repair of lesions made by activation-induced cytidine deaminase (AID) in immunoglobulin (Ig) variable region (V) genes in B cells is known to cause somatic hypermutation (SHM) of the antibody genes and, when coupled with selection mechanisms in germinal centers (GCs), leads to increased affinity of antibody. AID also induces double-strand breaks in switch region DNA that are necessary for antibody isotype switching. These processes are essential for maturation of the antibody response and defects in this pathway cause immunodeficiency syndromes. However, AID-induced lesions also cause genome-wide mutations and breaks that are associated with chromosomal translocations and lymphomagenesis. The cause of error-prone repair is unknown, and is one of the key remaining questions in AID biology. We recently discovered a dramatic decrease in the expression of an essential DNA repair protein, AP endonuclease 1 (APE1), in mouse GC B cells, and propose experiments that will explore whether this is the cause of error-prone repair and SHM at the Ig locus. APE1 is replaced in the germinal center by an inefficient homologue, APE2, about which very little is known. APE2 could recruit error-prone polymerases through its interaction with PCNA. I will test the hypothesis that differential expression of APE homologues in germinal centers is a major reason for error-prone repair of AID-induced lesions. In contrast to GC B cells, activated, cultured B cells do not undergo SHM and highly express APE1, which may promote accurate repair of AID-induced mutations in the V genes. By genetically reducing the expression of APE1, coupled with retroviral over-expression of APE2, we have developed a novel system that mimics ratios of APE1:APE2 protein in the GC, and can now detect AID-induced VDJH4 mutations in primary B cell cultures for the first time. Our preliminary data support that we are detecting bona fide SHM, and I propose to further optimize this system using VHb1-8 knock-in mice crossed to our mice with reduced APE1 and no APE2 (apex1+/-apex2Y/-) and analyzing mutations by deep sequencing. I expect mutation frequencies to be even higher in the VDJ region allowing us to analyze the spectrum of mutations. I will analyze functional domains of APE1 and APE2 that likely promote error-free vs. error-prone repair, and identify proteins that interact with APE1 and APE2 in B cells by co-immunoprecipitation and mass spectroscopy, which I expect will provide new insight into error-prone repair. In Aim 2 I propose to enforce expression of APE1 in the GC in vivo by introducing a transgene into mice that will express APE1, contingent upon AID, since AID is only expressed in activated and GC B cells. We will examine the effect of enforced APE1 expression on SHM. I expect these experiments will show that low expression of APE1 is the cause of error-prone repair of AID lesions in GCs.